Gene carrier using cell-derived nanovesicles and method for preparing the same

a cell-derived nanovesicle and gene carrier technology, applied in the direction of microcapsules, active genetic ingredients, gene material ingredients, etc., can solve the problems of high efficacy, safety of a gene therapeutic agent, and extremely rare that they are actually commercialized, and achieve the effect of efficient introduction into the target cell

Active Publication Date: 2017-10-26
IND ACADEMIC CORP FOUND YONSEI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

The present invention provides a method for efficiently introducing genes into target cells using cell-derived nanovesicles. This is accomplished by preparing nanovesicles through the outbudding of vesicles from cells and then inserting nucleotides into them. The resulting gene carrier can be used as a component for gene therapeutic agents or cell therapeutic agents. Overall, the invention improves the efficiency and safety of gene therapy and cell therapy methods.

Problems solved by technology

However, even with such attention, gene therapeutic agents have not shown high efficacy in early clinical trials, and thus it is extremely rare that they are actually commercialized.
Main causes for this are safety of a gene therapeutic agent and low gene delivery efficiency to a target cell.
Today, while viral gene carriers with high delivery efficiency have been most widely used, viral vectors such as a retrovirus, an adenovirus, and an adeno-associated virus have many limitations in being applied to the human body because of a complicated production process, safety problems such as immunogenicity, infection potential, inflammation induction and non-specific DNA insertion, and a limited size of acceptable nucleic acids.
However, these carriers also have disadvantages in that they have low blood stability, low gene delivery efficiency and low competitive prices.

Method used

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  • Gene carrier using cell-derived nanovesicles and method for preparing the same
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  • Gene carrier using cell-derived nanovesicles and method for preparing the same

Examples

Experimental program
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preparation examples

Preparation Example 1. Preparation of Gene Carrier Based on Cell-Derived Nanovesicles

[0056]1-1. Preparation of Gene Carrier for RNA Delivery

[0057]In a HEPES buffer (10 mM HEPES, 150 mM NaCl, 2 mM CaCl2, pH 7.4) containing 25 mM parformaldehyde (PFA) and 2 mM dithiothreitol (DTT), a HEK-293 cell line was incubated at 37° C. for 3 to 4 hours to artificially outbud vesicles from the cells, and the culture dish was shaken every 30 minutes to obtain a higher yield of vesicles. Subsequently, the resulting suspension containing the vesicles was sequentially subjected to centrifugation, sonication and extrusion, thereby preparing cell-derived nanovesicles with a uniform size. Afterward, a nucleic acid molecule composed of RNA was introduced into the nanovesicles using electroporation, resulting in the preparation of a gene carrier for RNA delivery.

[0058]1-2. Production of Gene Carrier for DNA Delivery

[0059]According to the same method as described in Preparation Example 1-1, a HEK-293 cell ...

experimental examples

Experimental Example 1. Analysis of Physical Properties of Nanovesicles

[0061]Exosomes are cell-derived natural substances, which have been known, according to a recent report, to be used as a gene carrier. Therefore, in this experimental example, physical properties of the nanovesicles prepared in Preparation Example 1-1 (plasma membrane-derived nanovesicles; PMNVs) were compared with those of the exosomes.

[0062]Specifically, a nanovesicle structure was observed using a transmission electron microscope (TEM), a zeta potential was measured by dynamic light scattering (DLS) and nanoparticle tracking analysis (NTA), and production per single cell and production per single cell per unit time were measured to compare productivities. In addition, fluorescence intensities were measured when Cy5-labeled siRNAs were delivered by simple mixing and by insertion, so as to verify that a gene carrier using nanovesicles or exosomes can be internalized in cells, and a quantity of siRNA inserted int...

experimental example 2

cle Yields and Gene Delivery Efficiency According to Treatment Condition

[0065]2-1. Comparison of Nanovesicle Yields According to PFA and DTT Treatment Conditions

[0066]In the preparation of the gene carrier of Preparation Example 1, a large quantity of vesicles could be artificially outbudded from cells by culturing the cells in a PFA and DTT-added HEPES buffer. In this experimental example, to deduce the optimal conditions for further enhancing the production yield of nanovesicles, various concentrations of PFA (5 mM, 25 mM, and 50 mM) or DTT (0.25 mM, 1 mM, and 2 mM) were added to a buffer solution, and the nanovesicles were prepared by the same method as described in Preparation Example 1-1 under conditions in which the DTT concentration was constantly maintained at 1 mM while the PFA concentration varied, and the PFA concentration was constantly maintained at 25 mM while the DTT concentration varied. Afterward, the nanovesicle yields per treatment concentration were quantitativel...

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Abstract

Provided are a gene carrier using cell-derived nanovesicles and a method for preparing the same. The gene carrier prepared by inserting a gene into the nanovesicles artificially outbudded from a plasma membrane has excellent delivery efficiency to a target organ and cells, induces long-term regulation of gene expression, and facilitates mass production due to a simple preparation process, and thus can be used as a core technique for the gene or cell therapeutic agent field.

Description

CROSS-REFERENCE TO RELATED APPLICATION[0001]This application claims priority to and the benefit of Korean Patent Application No. 2016-0049854, filed on Apr. 25, 2016, the disclosure of which is incorporated herein by reference in its entirety.BACKGROUND1. Field of the Invention[0002]The present invention relates to a gene carrier using cell-derived nanovesicles and a method for preparing the same.2. Discussion of Related Art[0003]Gene therapy is a treatment of a disease by delivering a therapeutic gene to a desired organ in the body to express a new protein in a cell, and recently, its valid therapeutic effects on various diseases such as viral diseases, cancer and the like have been widely reported. As the initial gene therapeutic agent has been commercially available in Europe, active studies on this are also progressing in Korea. Actually, some gene therapeutic agents show excellent therapeutic effects in clinical trials, and therefore expectations are high for the development of...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): A61K9/51A61K31/713A61K48/00
CPCA61K9/51A61K9/5192A61K31/713A61K48/005A61K9/5068C12N15/111C12N15/88C12N2320/32B82Y5/00C12N15/87
Inventor CHO, SEUNG-WOOJIN, YOON HEE
Owner IND ACADEMIC CORP FOUND YONSEI UNIV
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